The overall goal of this work is to develop anatomical, functional, and molecular magnetic resonance imaging (MRI) techniques that allow non-invasive assessment of brain function and apply these tools to study plasticity and learning in the rodent brain. MRI techniques are having a broad impact on understanding brain. Anatomical based MRI has been very useful for separating gray and white matter and detecting numerous brain disorders. Functional MRI techniques enable detection of regions of the brain that are active during a task. Molecular MRI is an emerging area, whose major goal is to image a large variety of processes in tissues. The goal of this project is to translate MRI developments in all these areas to study system level changes that occur in the rodent brain during plasticity and learning. Aim 1: Over the past few years, we have completed studies in the rodent brain that acquired very high temporal and spatial resolution functional MRI (fMRI) to monitor changes in hemodynamics as a surrogate marker of electrical activity during forepaw stimulation. Over the past couple of years we have demonstrated that fMRI from single venuoles can be detected with BOLD fMRI. Over the past two years we have completed a publication that demonstrates that single arterioles from deep cortex can be effectively imaged using blood volume based MRI techniques. Consistent with our understanding arteriole blood volume responds earlier that venous BOLD and the two vessel populations can be separated based on time to peak response. In blood volume the arteriole contribution dominates as compared to the tissue or venuole contributions. For BOLD the venuole contribution dominates compared to the arteriole contribution. Over the past year we have begun to establish the histological tools that will allow us to verify our MRI assignment of vessels and determining what fraction of arterioles and venuoles can be detected. Over the past few years we have demonstrated that the onset of fMRI enables extracting information about the laminar onset of neural activity in a brain region. We have continued to asses the determinants of the early hemodynamic response and in particular the role of pericytes in initiating the early response. An alternative hypothesis that more co-ordinated cellular activity occurs is also being pursued. Aim 2: Over the past several years we have demonstrated that manganese chloride enables MRI contrast that defines neural architecture, can monitor activity, and can be used to trace neural connections. We completed a publication that shows that detecting olfactory bulb layers is a sensitive way to monitor neurodegeneraton in a mouse model of app induced olfactory bulb degeneration. This demonstrates the MRI at this very high level of resolution can detect layer specific degeneration. A study is being completed for publication that uses a hippocampal slice preparation to study the synaptic mechanism underlying the MRI properties of manganese. Results indicate that a number of transport processes influence the accumulation and transport of Mn in these slice cultures. This indicates that the slice cultures in combination with MRI will be a very useful platform for understanding the basis for MRI contrast. Finally, we have continued a collaboration with Dorian McGavern, where we have shown that Mn can enter the brain through the skull at specific locations and once inside the brain trace appropriate neural connections. A second publication describing the preference for manganese to traverse areas of the brain dense in vessels and marrow has been published. Work this year has been aimed at characterizing the distribution of rodent skull vessels and marrow and begin to selectively use this system for delivery to the brain. Aim 3: Over the past few years we established a rodent model that uses peripheral denervation to study brain plasticity in response to the injury. Over the past couple of years we have shown that denervation of the infraorbital nerve leads to large increases in barrel cortex responses along the spared whisker pathway as well as large ipsilateral cortical activity consistent with our previous work in the forepaw and hindpaw. fMRI and manganese enhanced MRI predicted a strengthening of thalamo-cortical input along the spared pathway which was verified in slice electrophysiology studies in collaboration with John Isaac. Prior to this it was widely believed that the thalamo-cortical input was not capable of strengthening after the critical period. We have publishes a paper this year that demonstrate the synaptic mechanisms of this strengthening. This work conclusively shows that the denervation is causing a re-activation of the ability of this synapse to demonstrate long term potentiation. This LTP is occurring via activation of silent synapses. Interestingly, LTP is not dependent on the NR2B subunit, but infusion of NR2B inhibitors into the rodent cortex indicate that NR2B is important for establishing silent synapses. We have shown that trimming whiskers has a similar effect as infraorbital nerve cut showing that down regulation of use is enough to cause this LTP in the good whisker pathway. Evidence from manganese based track tracing shows a strengthening of input into layer 2/3 and 5 from the contralateral cortex to the ipsilateral cortex from the healthy whiskers. This year we have completed a study that demonstrates in slice the synaptic basis for the changes in the callosal input. We have demonstrated that this input can undergo LTP in the adult and that the callosal inputs are strengthened on to layer 5 pyramidal neurons. This strengthening is so large that this synapse can no longer undergo LTP. Studies are underway to determine if the potential for LTP is still present in these cells or whether there has been a change that makes it no longer possible for LTP. In both cases it is clear that large changes in the input strength are contributing to the adult plasticity detected. Aim 4: Progress in this aim over the past year has been slow due to lack of a specific person to carry out the work. Fortunately a new member of the laboratory has reactivated this exciting project. We have re-established our ability to do fear condition experiments. We are validating that earlier Mn tracing experiments that indicated plasticity at novel synapses during fear conditioning are reproducible.. If so we will take the approach as Aim 3 to determine the synaptic basis for the changes using slice electrophysiology.